Dystroglycan is an integral membrane protein that links the cytoskeleton and extracellular matrix (Curr. Opin. Cell Biol. 8: 625-631 (1996)). Although first discovered in skeletal muscle as a component of the dystrophin-glycoprotein complex, it has now become clear that dystroglycan is expressed in many cell types and tissues. An underlying theme in the expression pattern of dystroglycan is that it is expressed in most cell types directly associated with basement membranes (e.g., muscle, peripheral nerve, and epithelia) (J. Histochem. Cytochem. 46: 449-457 (1997)). Dystroglycan null mouse embryos fail to progress beyond the early egg cylinder stage (5.5 days) and manifest structural and functional defects in Reichert""s membrane, one of the first basement membranes that form in the rodent embryo (Hum. Mol. Genet. 6: 831-841 (1997)). Moreover, dystroglycan has recently been shown to be required for the formation of basement membranes in embryoid bodies, a system that models early embryonic development (Cell 95: 859-870 (1998)). Dystroglycan is also required for the organization of laminin, a key extracellular matrix ligand for dystroglycan, on cell surfaces (Cell 95: 859-870 (1998)). Taken together these observations suggest a model whereby dystroglycan organizes laminin, and perhaps other matrix molecules, on the cell surface in a manner that is important for the formation of basement membranes at the tissue level of organization.
Longstanding observations in the field of cancer biology indicate that transformed cells exhibit numerous aberrant interactions with the extracellular matrix in general and more specifically with basement membranes. For example, transformed cells typically exhibit a marked reduction of fibronectin (Cell 11: 115-126 (1978)) and laminin (J. Cell. Biol. 88: 352-357 (1981)) associated with their surfaces, compared to non-transformed controls. Moreover, invasive or metastatic cancers of epithelial origin often exhibit gross disruptions in the integrity of their underlying basement membranes (Lab Invest. 49: 140-147 (1983)). This is thought to reflect the role of basement membranes as a physical barrier between the epithelium and stroma, and the necessity that this barrier be breached in order for epithelioid cancer cells to invade the stroma as they metastasize to distant sites. On the other hand, many basement membrane proteins may also provide positive trophic support for transformed cells, and in some types of neoplastic disease, invasive lesions may retain their association with basement membrane proteins (Lab Invest. 49: 140-147 (1983)). These behaviors of tumor cells are quite complex and involve numerous mechanisms including secretion of matrix degrading enzymes and altered expression of matrix proteins, and their receptors, on the cell surface (Crit. Rev. Oncol. Hematol. 26: 163-173 (1997)). The demonstrated ability of dystroglycan to bind and organize laminin on the cell surface, and its requirement for the de novo assembly of basement membranes, suggests that dystroglycan might be involved in cancer cell invasion and metastasis. One possibility is that a reduction or ablation of dystroglycan expression in transformed cells may alter cell interactions (e.g., anchorage) with the extracellular matrix, or alter the integrity of cell basement membranes in such as way as to promote the ability to invade tissues or metastasize.
The present invention relates in one aspect, to a method for diagnosing the tumorigenic grade of a malignant tissue, the method comprising determining the amount of dystroglycan protein of the malignant tissue relative to a standard. In one embodiment, the amount of dystroglycan protein of the tissue is determined by measuring the amount of mRNA transcripts which encode dystroglycan, accomplished for example by northern blot analysis of the RNA in the tissue or polymerase chain reaction to specifically amplify dystroglycan mRNA transcripts or a segment thereof. In another embodiment, the amount of dystroglycan protein in the tissue is determined by performing western blot analysis or immunofluorescence analysis on the tissue components to detect xcex1-dystroglycan or xcex2-dystroglycan. An antibody probe suitable for such analyses, which binds specifically to the C-terminus of xcex2-dystroglycan, is provided. This method is applicable to human malignant tissue, especially adenocarcinoma, and preferably prostate or mammary adenocarcinoma.
In another aspect, the present invention relates to a method for determining the prognosis of a patient afflicted with a malignancy, the method comprising determining the expression level of the dystroglycan gene in a tissue sample of the malignancy, and comparing the expression level to a standard, with a decreased level of dystroglycan expression being indicative of unfavorable prognosis. The expression level of the dystroglycan gene may be determined by determining the amount of mRNA transcripts of the gene or by determining the level of the dystroglycan protein relative to a standard amount of said transcripts or protein. This method can also be applied to detection of a cancerous disease state in a tissue of a patient, a decreased level of dystroglycan protein being indicative of the presence of cancer.
In another aspect, the present invention relates to a method for identifying an individual at risk for the development of cancer, or an individual at risk for the recurrence of cancer after treatment, the method comprising determining the level of expression of the dystroglycan gene in tissue samples from an individual, and comparing the dystroglycan expression level in the sampled tissue with a normal dystroglycan expression level, a decreased level of dystroglycan expression being indicative of high risk for development or recurrence of cancer.
In another aspect, the present invention relates to a method for identifying individuals at risk for developing cancer comprising, screening for mutations in the dystroglycan genes of the individual. One such mutation is the allelic loss of human chromosome 3p21. The mutations may be identified by amplifying coding regions of the dystroglycan gene of the individual by polymerase chain reaction, and then examining the amplified regions for mutations which affect the function of the dystroglycan protein product.